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Home , Enzyme inhibitor, Tranylcypromine

Insights into the mode of inhibition of human mitochondrial monoamine B from high-resolution crystal structures

Claudia Binda*, Min Li†, Frantisek Huba´ lek†, Nadia Restelli*, Dale E. Edmondson†‡, and Andrea Mattevi*‡

*Department of Genetics and Microbiology, University of Pavia, 27100 Pavia, Italy; and †Departments of and Chemistry, Emory University, Atlanta, GA 30322

Communicated by Judith P. Klinman, University of California, Berkeley, CA, June 23, 2003 (received for review April 11, 2003) B (MAO-B) is an outer mitochondrial mem- We report here the structures of MAO-B in complex with brane-bound that catalyzes the oxidative deamination several reversible and irreversible inhibitors (Fig. 2) to elucidate of arylalkylamine neurotransmitters and has been a target for a their respective binding modes as well as to provide insights into number of clinically used inhibitors. The 1.7-Å structure of the mode of inhibition. Higher (1.7 Å) resolution data were the reversible isatin–MAO-B complex has been determined; it obtained that provide additional structural details on the ac- forms a basis for the interpretation of the enzyme’s structure tive site relevant to and to the detailed catalytic when bound to either reversible or irreversible inhibitors. 1,4- mechanism. Diphenyl-2-butene is found to be a reversible MAO-B inhibitor, The structure of MAO-B in complex with isatin was deter- which occupies both the entrance and cavity space in mined because this compound is found at higher levels in the enzyme. Comparison of these two structures identifies patients with neuropathological conditions and has been shown ␮ Ile-199 as a ‘‘gate’’ between the two cavities. Rotation of the to be a competitive MAO-B inhibitor with a Ki of 3 M (2). In side chain allows for either separation or fusion of the two the course of these studies, we have uncovered a competitive ϭ ␮ cavities. Inhibition of the enzyme with N-(2-aminoethyl)-p-chlo- MAO-B inhibitor (Ki 35 M) that is identified as 1,4-diphenyl- robenzamide results in the formation of a covalent N(5) flavin 2-butene, a contaminant of polystyrene products (3). adduct with the phenyl ring of the inhibitor occupying a position The structure of MAO-B after inhibition with N-(2- in the catalytic site overlapping that of isatin. Inhibition of aminoethyl)-p-chlorobenzamide (also known as Ro 16-6491) was MAO-B with the clinically used trans-2-phenylcyclopropylamine elucidated. This compound belongs to a class of highly specific results in the formation of a covalent C(4a) flavin adduct with an MAO-B inhibitors, which includes . A previous study opened cyclopropyl ring and the phenyl ring in a parallel (4) suggested this inhibitor binds to a site on the enzyme other orientation to the flavin. The bond between the flavin- than the flavin moiety. (trans-2-phenylcyclo- substituted Cys-397 and Tyr-398 is in a cis conformation, which ) has been known for Ͼ40 years as a MAO inhibitor allows the proper orientation of the phenolic ring of Tyr-398 in and used clinically as an . As with the lazabemide the . The flavin ring exists in a twisted nonplanar class of inhibitors, tranylcypromine has been thought to exert its conformation, which is observed in the oxidized form as well as inhibitory action as a mechanism-based inhibitor by covalent in both the N(5) and the C(4a) adducts. An immobile water binding of the MAO-B-catalyzed reaction to an amino is H-bonded to Lys-296 and to the N(5) of the flavin as acid side chain (5, 6). The structural data presented in this study observed in other flavin-dependent amine . The active show that, in contrast to previous conclusions, tranylcypromine site cavities are highly apolar; however, hydrophilic areas exist and N-(2-aminoethyl)-p-chlorobenzamide inhibit MAO-B by near the flavin and direct the amine moiety of the substrate for covalent binding to the flavin ring, however, at differing sites on binding and . Small conformational changes are ob- the isoalloxazine ring. served on comparison of the different inhibitor–enzyme com- plexes. Future MAO-B drug design will need to consider ‘‘in- Materials and Methods duced fit’’ contributions as an element in –enzyme Expression, purification, absorption spectral studies, and cata- interactions. lytic assays of recombinant human liver MAO-B were performed as described (7). All chemicals used in this study were obtained he structure and function of monoamine oxidases A and B from commercial sources. T(MAO-A and -B) have been of interest to a wide variety of Orthorhombic MAO-B crystals were grown by the sitting-drop scientific disciplines because of the role of these in the vapor diffusion method at 4°C (1) using polystyrene micro- oxidation of arylalkylamine neurotransmitters such as bridges obtained from Hampton Research. The precipitant and . The proposed role of MAO-B in age-dependent solution consisted of 12% (wt͞vol) PEG 4000, 70 mM neurodegenerative diseases has resulted in a renewed interest in sulfate, and 100 mM N-(2-acetamido)-2-iminodiacetic acid this enzyme as a target for the development of neuroprotective (ADA), pH 6.5. The solution contained 2 mg of MAO-B agents. MAO-B inhibitors are used clinically and others are in per ml, 8.5 mM Zwittergent 3–12, and 25 mM potassium development. MAO-A and -B have been extensively investigated phosphate buffer, pH 7.5. Crystals of enzyme–inhibitor com- and serve as the prototype for the flavin-dependent amine plexes were obtained by incubating the protein with 2 mM oxidases. inhibitor followed by gel filtration on Superdex 200. For all The recent description of the 3.0-Å structure of human recombinant MAO-B in its -inhibited form by our laboratories (1) revealed a two-domain architecture of the Abbreviation: MAO, monoamine oxidase. molecule and its mode of binding to the mitochondrial outer Data deposition: The atomic coordinates and the structure factors have been deposited in the Protein Data Bank, www.rcsb.org (PBD ID codes 1OJA, 1OJB, 1OJC, 1OJD, and 1OJ9). membrane through a C-terminal hydrophobic ␣-helix. These ‡To whom correspondence may be addressed at: (D.E.E.) Department of Biochemistry, studies show the substrate negotiates a protein loop in its entry Emory University School of Medicine, 1510 Clifton Road, Atlanta, GA 30322. E-mail: into the active site of the enzyme, which involves traversing an [email protected]; or (A.M.) Department of Genetics and Microbiology, ‘‘entrance cavity’’ before entering the ‘‘substrate cavity’’ (Fig. 1). University of Pavia, Via Abbiategrasso, 27100 Pavia, Italy. E-mail: [email protected].

9750–9755 ͉ PNAS ͉ August 19, 2003 ͉ vol. 100 ͉ no. 17 www.pnas.org͞cgi͞doi͞10.1073͞pnas.1633804100 Downloaded by guest on September 30, 2021 atomic models were built with program O (12). Unbiased Ϫ Ϫ 2Fo Fc and Fo Fc maps were used to model the inhibitors which, in all cases, were well defined by the electron density maps (Fig. 3). The models exhibit good stereochemical pa- rameters with only three residues in each monomer (Lys-52, Ala 346, and Asp 419) in disallowed regions of the Ramachan- dran plot. The refinement statistics are listed in Table 1. Analysis of the final models were carried out with the pro- grams GRID (13) and VOIDOO (14). Pictures were produced by using LIGPLOT (15), BOBSCRIPT (16), MOLSCRIPT (17), and RASTER3D (18). Results and Discussion Structural Analysis of Reversible, Noncovalent Inhibitor–MAO-B Com- plexes. The tight, reversible binding of isatin (Fig. 2) to MAO-B has been known since its original identification (2). Isatin competitively binds to purified, recombinant MAO-B with a Ki of Ϸ3 ␮M. The tight binding of the inhibitor led to an improved crystal quality and diffraction data could be measured up to Fig. 1. Overall three-dimensional structure of human MAO-B monomeric 1.7-Å resolution (Table 1). A stereoview of this structure is unit in complex with 1,4-diphenyl-2-butene. The FAD-binding domain (resi- dues 4–79, 211–285, and 391–453) is in blue, the substrate-binding domain shown in Fig. 3. The electron density of the dioxoindole ring (residues 80–210, 286–390, and 454–488) is in red, and the C-terminal mem- shows its orientation in the substrate cavity to be perpendic- brane-binding region (residues 489–500) is in green. The FAD and the ular to the flavin ring with the oxo groups on the pyrrole ring inhibitor are shown as yellow and black ball-and-stick models, respectively. pointing toward the flavin. The 2-oxo group and the pyrrole The inhibitor binds in a cavity (shown as a cyan surface) that results from the NH are H-bonded to ordered water present in the fusion of the entrance and substrate cavities (see text). active site, whereas the 3-oxo function is not involved in any H-bond. The balance of the binding interactions involve many

van der Waals contacts between the isatin ring and BIOCHEMISTRY inhibitors except tranylcypromine, 2 mM inhibitor was added to residues in the solvent-inaccessible, hydrophobic substrate the crystallization drop. Triclinic crystals were grown as de- cavity. scribed (1). X-ray diffraction data were collected at 100 K at the An unanticipated noncovalent inhibitor of MAO-B was ser- Swiss Light Source in Villigen and at the beam-line ID14-EH4 endipitously discovered when we attempted to determine the of the European Synchrotron Radiation Facility in Grenoble, structure of the enzyme in the presence of d-. France. The high brilliance provided by these beam-lines re- Rather than containing amphetamine, the crystals were found to sulted in a significant increase of the resolution of the diffraction contain 1,4-diphenyl-2-butene (whose identification will be de- data. The diffraction images were processed with MOSFLM (8) tailed in a separate communication, ref. 3). The source of this and programs of the CCP4 package (9). compound was found to be an impurity released by the polysty- Structure refinements were performed with the programs rene microbridges used for crystallization of the enzyme. We Ϸ REFMAC5 (10) and WARP (11). Tight noncrystallographic sym- find that this compound competitively binds to MAO-B (Ki 35 metry restraints were applied throughout the refinements. The ␮M). Its presence does not interfere with structure determina- tions of MAO-B in complex with other inhibitors that have higher binding affinities or are covalently bound. The novel aspect of this inhibitor is that it is bound in a manner where one phenyl ring is in the entrance cavity space whereas the other phenyl ring is in the substrate cavity space (Figs. 1 and 4) overlapping the position occupied by isatin. The planes of the two phenyl rings of this inhibitor are orthogonal to one another. In this structure, the two cavities are now fused into one single cavity, which constitutes a significant proportion of the volume of the protein (Fig. 1). Comparison of the isatin and 1,4- diphenyl-2-butene structures shows that the side chain of Ile-199 exhibits differing rotamer conformations that function as a ‘‘gate’’ between the entrance and substrate cavities (Fig. 5). When isatin is bound, the Ile-199 side chain separates the two cavities. When 1,4-diphenyl-2-butene is bound, the side chain is rotated to a conformation such that the two cavities are no longer separated and are now fused forming a single cavity. This finding of an MAO-B inhibitor that spans both cavities in binding demonstrates that new reversible inhibitors could be developed that would use both cavities as potential binding targets. Un- published data in our laboratories have shown that trans-trans- 1,4-diphenyl-1,3-butadiene binds to MAO-B with a higher af- ϭ ␮ finity (Ki 7 M). These diphenyl compounds exhibit high specificity for MAO-B (103 higher affinity for MAO-B compared with MAO-A) and therefore could represent lead compounds for the development of MAO-B inhibitors. Fig. 2. Structures of MAO-B inhibitors used in this study and atomic num- The concept of cavity-spanning ligands is further supported by bering of the flavin ring. analysis of the triclinic MAO-B crystals that were obtained by

Binda et al. PNAS ͉ August 19, 2003 ͉ vol. 100 ͉ no. 17 ͉ 9751 Downloaded by guest on September 30, 2021 Fig. 3. Stereoview of the isatin and of the 2Fo Ϫ Fc electron density map calculated at 1.7-Å resolution and contoured at 1-␴ level. Carbons are in black, nitrogens in blue, oxygens in red, and sulfurs in yellow. H-bonds involving inhibitor atoms are outlined by dashed lines.

using the detergent lauryldimethylamine N-oxide and used for the inhibition of MAO-B with 2-phenylcyclopropylamine con- the original structure determination (Table 1) (1). Refinement cluded that the mode of inhibition was the binding of the of this second MAO-B crystal form (P1 symmetry with five oxidation product of MAO-B catalysis to Cys-365 (5, 6). dimers per unit ) shows the presence of a detergent molecule These studies, as well as spectral studies in our laboratory, bound to each enzyme monomer. The N-oxide end of the show that inactivation of MAO-B by either inhibitor leads to a molecule is positioned near the flavin and the aliphatic side stable bleaching of the flavin absorption spectrum even in the chain traverses the space formerly defined by the substrate and presence of oxygen. However, in either case, such putative flavin entrance cavities (data not shown). adducts are expected to be labile because denaturation of either inhibited enzyme results in the reappearance of a spectrum Structures of Inhibitor-Adducts That Result in a ‘‘Bleached’’ Flavin characteristic of oxidized flavin. Flavin bleaching is also ob- Spectrum. Two inhibitor classes of MAO-B that have attracted a served in the crystalline inhibited enzyme as measured by good deal of attention include the N-(2-aminoethyl)arylcarbox- single-crystal microspectrophotometry (data not shown). amides and trans-2-phenylcyclopropylamine (tranylcypromine). The structure of MAO-B after inhibition with N-(2- Previous studies on the inhibition of MAO-B by lazabemide or aminoethyl)-4-chlorobenzamide shows that the inhibitor is N-(2-aminoethyl)-4-chlorobenzamide suggest they are oxidized bound to the enzyme as a flavin N(5) adduct (Fig. 6). The by MAO-B to a form that binds to an active-site amino acid side aromatic ring of the inhibitor adopts the same orientation and chain (possibly histidine) in a labile bond that is reduced to a position as isatin. The resolution of the x-ray data cannot stable bond by treatment with cyanoborohydride (4). Studies of unambiguously determine the detailed molecular formula of this

Table 1. Data collection and refinement statistics N-(2-aminoethyl)-p- Lauryldimethyl- 1,4-Diphenyl-2-butene Isatin Tranylcypromine chlorobenzamide amine N-oxide

Resolution, Å 2.3 1.7 2.2 2.4 3.1 Space group C222 C222 C222 C222 P1 Cell, Å 131.1, 224.0, 86.8 130.3, 222.0, 86.0 130.6, 222.5, 86.2 131.7, 223.1, 86.5 108.4, 132.4, 154.8 ° 90.0, 90.0, 90.0 90.0, 90.0, 90.0 90.0, 90.0, 90.0 90.0, 90.0, 90.0 90.1, 90.5, 114.0 Unique reflections 49,954 128,815 64,040 47,190 140,447 Completeness, %* 90.6 (66.7) 94.4 (76.7) 99.9 (99.9) 94.5 (85.3) 98.4 (97.8) Redundancy 5.8 4.3 3.7 3.0 3.8 † Rsym* 0.107 (0.202) 0.083 (0.339) 0.130 (0.472) 0.096 (0.303) 0.088 (0.201) ‡ Rcryst 0.212 0.181 0.206 0.197 0.252 ‡ Rfree 0.253 0.204 0.247 0.250 0.260 rmsd bond angle, ° 1.2 1.4 1.1 1.1 1.5 rmsd bond length, Å 0.009 0.012 0.007 0.008 0.016 Number of atoms͞average B factor, Å2 Protein ϩ FAD 8,017͞43.7 8,017͞15.5 8,017͞45.4 8,017͞19.2 40,139͞40.7 Ligand 2 ϫ 16͞60.1 2 ϫ 11͞17.9 2 ϫ 10͞55.1 2 ϫ 13͞22.9 10 ϫ 16͞35.1 Water molecules 230͞39.5 661͞27.2 404͞29.4 418͞21.2 —

rmsd, rms deviation. *Values in parentheses are for reflections in the highest-resolution shell. † Rsym ϭ⌺͉Ii Ϫ ͗I͉͘͞⌺Ii, where Ii is the intensity of ith observation and ͗I͘ is the mean intensity of the reflection. ‡ Rcryst ϭ⌺͉Fobs Ϫ Fcalc͉͞⌺͉Fobs͉, where Fobs and Fcalc are the observed and calculated structure factor amplitudes, respectively. Rcryst and Rfree were calculated by using the working and test set, respectively.

9752 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.1633804100 Binda et al. Downloaded by guest on September 30, 2021 Fig. 5. Isocontour of GRID molecular interaction fields computed with an 2 aromatic sp carbon probe (gray) and with a neutral NH2 probe (cyan). The contour encloses points with negative values under Ϫ2 kcal͞mol for the sp2 carbon probe and Ϫ5 kcal͞mol for the NH2 probe. This picture was generated by using the coordinates from the 1,4-diphenyl-2-butene complex with MAO-B. The inhibitor is highlighted in dark gray ball-and-stick; the inhibitor ring in contact with Phe-103, Ile-199, and Ile-316 occupies the entrance cavity space, whereas the inhibitor ring in contact with Tyr-398 and Tyr-435 occupies the substrate cavity space (Fig. 1). Atoms are colored as in Fig. 3. The confor- BIOCHEMISTRY Fig. 4. LIGPLOT (15) illustration of 1,4-diphenyl-2-butene binding to MAO-B. mation of Ile-199 as observed in the isatin complex is shown in red to depict Dashed lines indicate H-bonds. Carbons are in black, nitrogens in blue, oxy- the in this residue that leads to merging of the two gens in red, and sulfurs in yellow. Water molecules are shown as cyan spheres. cavities, which is observed in the MAO-B complexes with N-(2-aminoethyl)-4- chlorobenzamide, 1,4-diphenyl-2-butene, or lauryldimethylamine N-oxide.

adduct, as shown in Fig. 7. However, preliminary mechanistic ␣ studies of this inhibition suggest the amine nitrogen is released the 8 -S-cysteinyl-FAD cofactor of MAO-B. In all structures, an before forming the N(5) adduct. The structural data show that ordered water molecule H-bonds to the N(5) of the flavin and ␧ the Ile-199 side chain is in the same rotamer conformation the -amino group of Lys-296, which defines a triad [Lys–H2O– exhibited by 1,4-diphenyl-2-butene such that the entrance and flavin N(5); Fig. 4] also found in the structures of other substrate cavities are fused. This change in conformation is flavin-dependent amine oxidases (20). A second point to be necessary to accommodate the p-chloro substituent on the made is that the of the FAD-linking cysteinyl benzamide ring. residue (Cys-397–Tyr-398) is in an energetically unfavorable cis Structural data of MAO-B inhibited by 2-phenylcyclopro- rather than trans conformation. Structural analysis of MAO-B shows that this cis Cys-397–Tyr-398 peptide bond results in a pylamine show that this inhibitor binds to the flavin as a C(4a) favorable steric orientation of the phenolic ring of Tyr-398, adduct (Fig. 6). The structure of this adduct differs from that which is a component of the active site (1). Examination of formed with N-(2-aminoethyl)-4-chlorobenzamide not only in structures of other flavoenzymes containing 8␣-covalent flavins the flavin ring substitution site but also in the orientation of the shows only trans conformations of the C-terminal peptide link- phenyl ring, which is parallel to the flavin ring rather than age of the residue covalently bound to the flavin. Therefore, this perpendicular as found in all other complexes. To accommodate Ϸ cis linkage appears to be unique to MAO-B rather than a general this ring orientation, Gln-206 must move 1 Å. The conforma- feature associated with covalent binding of flavin coenzymes to tion of the phenyl ring of the tranylcypromine complex does not . cis Nonproline conformers are often found proximal to extend in the substrate cavity far enough to induce the rotamer the functional sites of proteins (21). This strained conformation conformation of Ile-199 to the open form. Thus, in this aspect, of the Cys-397–Tyr-398 peptide bond in MAO-B adheres to that the tranylcypromine-inhibited form resembles that of isatin- paradigm. inhibited MAO-B. Another source of strain about the flavin site is the bent and The electron density of the tranylcypromine–MAO-B com- twisted conformation of the normally flat aromatic isoalloxazine plex is best fit with a structure in which the cyclopropyl ring is ring of the flavin cofactor (Fig. 6). The angle between the open (Figs. 6 and 7). Formation of this flavin C(4a) adduct can dimethylbenzene ring and the ring is Ϸ30°. Similarly be accommodated by a mechanism for inhibition similar to that distorted flavin conformations have been found in other flavin- shown by Sayre’s laboratory for quinone-mediated oxidative dependent amine oxidases (20). Structural comparisons of the cleavage of cyclopropylamines (19). No extra electron density unsubstituted flavin ring with those of the N(5) and C(4a) due to inhibitor binding was observed around Cys-365 (see ref. adducts of inhibited MAO-B show the flavin ring to exhibit 5) or other sites on the protein. Further work is required for identical conformations. The nonplanar conformation of the verification of this proposed mechanistic pathway for MAO-B oxidized flavin ring structure is expected to facilitate the for- inhibition. mation of such adducts because of making the N(5) and C(4a) atoms more sp3-like in conformation. It is of interest that Structural Insights into the Flavin Cofactor of MAO-B. The increased MAO-B does not conform to the paradigm that flavoprotein resolution of the x-ray data permits some structural insights for oxidases readily form flavin N(5) sulfite adducts. The hydro-

Binda et al. PNAS ͉ August 19, 2003 ͉ vol. 100 ͉ no. 17 ͉ 9753 Downloaded by guest on September 30, 2021 Fig. 6. Crystallographic data of the flavin conformations in MAO-B and in the N(5) and C(4a) adducts. This stereo picture was produced by orienting the flavin ring perpendicular to the plane of the drawing. The electron density (1.7-Å resolution) for the covalently bound flavin in the structure of the isatin complex is shown at Top. The Middle structure shows the electron density for the covalent adduct with N-(2-aminoethyl)-p-chlorobenzamide (2.4 Å), and the Bottom structure is that formed with trans-2-phenylcyclopropylamine (2.2 Å). The contour level is 1 ␴.

phobic nature of the entrance and substrate cavities provides a flavin and is required for recognition and directionality of the structural rationale for this property. The energetics of an substrate amine functionality. This hydrophilic region is located anionic species traversing these hydrophobic cavities are simply between Tyr-398 and Tyr-435, which, together with the flavin, too formidable to overcome even though the flavin ring is in a form an aromatic cage for amine recognition (1, 20). Mutagen- conformation that readily would form such an adduct. esis studies targeting these Tyr side chains in both MAO-A and MAO-B support the key role of these residues in substrate Conclusions Regarding the Active-Site Structure of MAO-B. By using binding (22). We find several ordered, captive water molecules the program GRID (13), a picture of the inhibitor binding site for that fill in vacant space in the inhibitor binding cavity. Therefore, 1,4-diphenyl-2-butene can be viewed in regards to hydrophobic- the design of future MAO-B inhibitors should consider hydro- ity and hydrophilicity. As seen in Fig. 5, the major part of the philic substituents that could displace these ordered waters, cavity is hydrophobic, which allows for the tight binding of apolar resulting in an increased affinity and specificity. Another aspect substrates and inhibitors. The only hydrophilic section is near the emerging from this work are the subtle and inhibitor-dependent changes in conformation of residues in the binding cavity of MAO-B. These results are consistent with conclusions reached from quantitative structure–function activity relationship stud- ies (23) and demonstrate that induced fit and active site plasticity are important components of MAO-B inhibition.

We thank the staff of the Swiss Light Source and European Synchrotron Radiation Facility for help during x-ray data collection, Ms. Milagros Aldeco for technical assistance with this project, and Dr. A. Mozzarelli (University of Parma) for his assistance with microspectrophotometry experiments. This work was supported by grants from the National Institute of General Medical Sciences (GM-29433), the Ministero dell’Istruzione Fig. 7. Proposed structures of the flavin adducts with the MAO-B-catalyzed dell’Universita´ e della Ricera (FIRB, COFIN03, and Legge 449͞97), and oxidation products of N-(2-aminoethyl)-p-chlorobenzamide (Left) and of the Agenzia Spaziale Italiana. A.M. and C.B. acknowledge support from a trans-2-phenylcyclopropylamine (Right). Pfizer Technology Development Support grant.

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Binda et al. PNAS ͉ August 19, 2003 ͉ vol. 100 ͉ no. 17 ͉ 9755 Downloaded by guest on September 30, 2021